Collagen is a major structural protein useful in reconstructive therapeutic procedures in humans. Collagens used for these purposes are generally prepared by isolating the material from tissues of farm animals such as cows or pigs. While such isolated collagen has been used with some success, it is essentially a protein foreign to the treated human being and immunogenic responses can be a problem. This problem has been minimized by treating the animal-derived collagen with proteolytic enzymes to decrease immunogenicity.
It is clear that it would be advantageous to supply human rather than bovine or porcine collagen for therapeutic purposes. The sources for purified human collagen are limited and the only reliable source is human placenta. Human collagen can be purified from human placenta as described in copending U.S. patent application Ser. No. 07/921,810 (Collagen Corporation). The placenta contains several types of collagers, most notably types I, III, IV, and V. The process of separating and purifying one type from the others is imperfect and results in a predominant type with small amounts of the other types. Production of purified collagen from placentas further necessitates additional processing steps to ensure that the resulting collagen product is free from human viruses such as hepatitis and HIV. In view of this, there have been attempts to prepare human collagen using recombinant techniques.
Expression of the human cartilage procollagen gene (Col2A1) in mouse 3T3 cells been reported (Ala-Kokko, L. et al., J Biol Chem (1991) 266:14175-14178). Olsen, A. S. et al. reported expression of a minigene version of the human pro.alpha.1 (I) collagen gene in mouse fibroblasts (Olsen, A. S. et al., J Biol Chem (1991) 266:1117-1121). Full-length human pro.alpha.2 (V) collagen cDNA in pro.alpha.2 (V)-deficient hamster cells was reported by Greenspan, D. S. et al., J Biol Chem (1989) 264:20683-20687; mouse fibroblasts have also been used to express the pro.alpha.1 (I) chain wherein the resulting expressed protein is complexed in the collagen triple helix with murine pro.alpha.2 (I) chains, as described by Schnieke et al., Proc Natl Acad Sci USA (1987) 84:8869-8873. Transgenic mice that were modified to contain a mutated form of the pro.alpha.1(I) gent were not viable after birth, according to a study by Stacey, A. et al. Nature (1988) 322:131-136. In addition, transgenic mice have been obtained that express a minigene version of the human gene for type I procollagen systemically (Khillan, J. S. et al., J Biol Chem (1991) 266:23373-23379); PCT application WO92/22333. These mice are useful as model systems for investigating bone diseases characterized by the modified collagen produced.
The production of recombinant human collagen is made troublesome by the necessity for a multiplicity of posttranslational enzymes which are generally believed to be present only in cells which natively produce collagen. At least eight such posttranslational enzymes are believed to be needed (Prockop et al., New England J Med (1984) 311:376-386). This has limited attempts at recombinant production to cells which natively produce this protein; this inevitably results in chimeric forms of the protein.
In order to avoid chimeric collagens which contain partly human and partly host mammalian cell chains in the triple helix, it might be possible to use human cells for this production. Even in this case, however, it is not possible to obtain collagen product of a particular type free of other collagen types. As further described below, the variety of collagens produced and their innate similarity makes homogeneous preparations from either native or recombinant sources which produce their own collagen impossible.
The present invention solves these problems by effecting the synthesis of human procollagen or collagen in cells which do not natively produce this protein, employing techniques established for the production of foreign proteins in mammalian milk, as described in the publications cited hereinbelow. The collagen of designated types is secreted into the milk either as procollagen or collagen, depending on the construction of the expression systems and accompanying recombinant enzyme production.